Bilirubin's Chemical Formula

Frank J. Dinan

Two students meet a professor who surprises them by telling them that a biochemically important molecule’s structure is incorrectly represented in the literature, textbooks, and a reference source. The students are challenged to find the nature of the structural errors and to correct them. The case’s content deals with geometric isomerism and tautomerism.

The Case

Erin and Angelo listened closely to their guest seminar speaker, Professor Rita Barth, as she concluded her talk. She had certainly lived up to her advanced billing. Her explanation of the importance of understanding medicine at a molecular level was filled with interesting and insightful examples. Erin and Angelo had taken both organic chemistry and biochemistry, and Professor Barth’s talk helped them to see how important the concepts from these courses were to understanding the chemistry and biochemistry that underlies medicine at a molecular level. Because they both planned to pursue careers in medicine, her presentation was of great interest to them.

At the conclusion of her talk, Professor Barth offered to speak with anyone who had questions or concerns. In addition to being extremely knowledgeable, she seemed friendly, so the two students decided to speak with her about their future plans.

Erin approached and said, “Hello, Professor Barth, my name is Erin and this is my friend Angelo. We enjoyed your talk a lot. We are both interested in chemistry and want to attend medical school. We were wondering if you might have a problem that we could help you with. We have both taken organic chemistry and biochemistry and would love to put some of the ideas from those courses to work on a real-life problem.”

“It’s a pleasure to meet you, Erin and Angelo. I am so glad you enjoyed my talk.”

“Your examples were great,” said Angelo. “They showed us how lots of the ideas from our chemistry and biochemistry courses are useful in medicine.”

“So, you two are interested in applying some of the concepts that you have learned in class to a real-life medical problem? That’s great. I do have a problem that I’d love to have you look into, and your background should allow you to do so. Would it be possible for you to meet me in my office at the University Hospital at 9:00 on Saturday morning to discuss it further?” Erin and Angelo eagerly agreed.

When the students arrived at the hospital on Saturday morning, Professor Barth greeted them warmly. Once they were seated in her office, each sipping a cup of coffee, she started to explain the problem she had mentioned. “You are probably aware,” she began, “that newborn infants are often jaundiced and require phototherapy treatment with visible light to overcome that condition and obtain their normal skin color.”

“I’ve heard about that,” said Erin, “but I don’t know anything about the cause of the jaundice or what the light treatment does to clear it up.”

“I’m not surprised that you don’t, Erin. Let me try to explain. The jaundice arises from the fact that hemoglobin present in the infant’s body undergoes decomposition to form a yellow-orange compound called bilirubin. If the newborn’s liver is immature, it can’t process and remove the bilirubin from its body as a fully developed liver would. The build-up of bilirubin in the infant’s body causes the yellow skin color that we call jaundice. If the bilirubin is not removed and becomes too concentrated, it can do serious, life-long harm to the infant.”

“How does the light treatment allow the body to get rid of the bilirubin?” asked Angelo.

“You are familiar with geometric isomers, aren’t you?” asked the professor.

“Sure,” said Erin. “We called them cis/trans and Z/E-isomers, too.”

“Right, ‘cis/trans isomers’ is the term used when there are like groups present on the adjacent carbon atoms of a double bond or ring, and ‘Z/E- isomers’ is used in more complex situations—when like groups aren’t present, and the simple cis/trans method can’t be used.”

“I remember that we had to assign priorities to the groups attached to the double bond so we could decide whether a compound was a Z- or an E-isomer,” said Angelo.

“Well, geometric isomerism has a lot to do with the answer to your question, Angelo. Radiating an infant with visible light (we call it phototherapy) provides the energy needed to allow the naturally occurring bilirubin isomer formed by the degradation of hemoglobin to change into a different geometric isomer. The second isomer is more soluble in bile, urine, and feces than the first, so it is more easily eliminated from the infant’s body.”

“I see,” said Erin. “Changing the bilirubin that forms in the infant’s body into a more soluble isomer allows it to be removed from the body without the liver having to do the whole job.”

“You’ve got it, and that brings us to the problem that you two could give me a hand with.”

“Sure, that’s what we’re here for. What can we do?” asked Angelo, eagerly.

“You can help sort out a lot of confusion that exists in the biochemical literature and in some of the best biochemistry textbooks about the structure of bilirubin and the geometry and nomenclature of its two geometric forms—the one formed from the degradation of hemoglobin, and the other more soluble isomer that forms after phototherapy.”

Professor Barth went to a filing cabinet and withdrew several sheets of paper. The students could see drawings of the structures of a number of heterocyclic organic molecules.

Pointing to the structures, Professor Barth said, “The structure of bilirubin was originally established by a Noble Laureate, Hans Fischer, in 1941 (Fischer, Pieninger, and Weiss-barth 1941). He knew that bilirubin could exist in geometric isomeric forms, and because he didn’t know whether the bilirubin that he had isolated existed as the Z,Z- or E,E-form or, perhaps, in some combination of these forms, he always drew it in a linear form like this, one that didn’t indicate the geometry about the double bonds” (Figure 1).

“Years later, one of Fischer’s students, Rudolph Lemberg, studied the naturally occurring form of bilirubin and claimed that it exists in the Z,Z- form (Lemberg and Legge 1949). If you look, for example, in the widely used biochemistry text by Lenninger (Nelson and Cox 2000), or in a very important recent paper in the biochemistry literature that establishes bilirubin as a key molecule in a biochemical cycle that protects us against free radicals (Baranano et al. 2002), you will find this structure drawn for bilirubin. It implies that bilirubin exists in the E,E-isomeric form.” Professor Barth pointed to another structure (Figure 2).

“And there’s also another problem with bilirubin’s representation in reference sources. The problem concerns tautomerism in the two terminal pyrrole rings of bilirubin. Here, let me show you what I mean.”

Professor Barth pointed to some additional structural formulas and said, “I’m sure that you are familiar with enol-keto tautomerism in carbonyl compounds, but you may be less aware that tautomers also exist for amide carbonyl groups. Amide tautomers involve the nitrogen atom and are called lactim and lactam forms.” She then showed them some additional structures (Figure 3).

“The problem is,” she continued, “that some texts show the two terminal pyrrole rings of bilirubin in the lactim form and others show them in the lactam form. For example, the 12th edition of The Merck Index shows bilirubin’s pyrrole rings in the lactim form (Budvari 1996), and you have already seen that Lenninger’s textbook shows them in their lactam form (Nelson and Cox 2000).”

Professor Barth showed Angelo and Erin a representation of bilirubin’s terminal pyrrole rings (Figure 4) that is similar to the one that appears in The Merck Index.

Erin said, “I’m surprised that this confusion exists, but what can we do?”

“Good question, Erin. I would like you two to double check the literature and find the correct structure for the bilirubin isomer that forms in the body when hemoglobin decomposes, and the structure of the bilirubin isomer that forms after phototherapy. Also, I’d like you to clear up the ambiguity about the lactim versus lactam forms for the pyrrole rings present in bilirubin.”

“We’d like to give it a try,” said Angelo.

“Great,” said Professor Barth. “When you have the structures figured out, please assign priorities to the substituents present on the bilirubin double bonds, and then draw correct structures for the two forms of bilirubin and label them as the Z,Z- and E,E-isomers.”

Erin asked, “When you say draw correct structures for bilirubin, do you mean structures that show the correct geometry about the double bonds?”

“Yes, Erin, that’s exactly what I mean. Also, be sure that you have the correct lactim or lactam form drawn for the terminal pyrrole rings.”

Angelo said, “We’ll get right at it, but could I ask what you’ll do with this information?”

“When you have done these things I will ask you to write a brief paper that I can use with my first-year medical students. By correctly explaining and identifying the bilirubin structures, your paper will be a big help to them. It will clear up the ambiguity that exists in the literature and will help their understanding of the hemoglobin-bilirubin-phototherapy process.”

“We’ll do our best, Professor Barth, and we’ll get back to you as soon as possible.”

“That’s great,” Professor Barth replied. “I will be looking forward to your findings.”

Findings and Assignment

After researching the primary literature, Erin and Angelo uncovered the following information about bilirubin:

• The decomposition of hemoglobin in the body results in the formation of the Z,Z-isomer of bilirubin.
• Phototherapy results in the conversion of the Z,Z-isomer of bilirubin into the E,E-isomer.
• The two terminal rings of bilirubin exist in the lactam, not the lactim form.

Based on these findings, students should complete each of the following tasks (answers are in Figure 5):

• Task 1. Assign Cahn-Ingold-Prelog priorities to each of the substituent groups attached to the double bonds substituted on the terminal pyrrole rings of bilirubin.
• Task 2. Use these priorities to draw geometrically correct structures for the Z,Z-isomer, the E,E-isomer, and an E,Z-isomer of bilirubin and label each.
• Task 3. Draw both the lactim and lactam forms for each of the terminal pyrrole rings of bilirubin and properly label each. Pay attention to the placement of the double bonds in the rings.
• Task 4. Suggest a reasonable mechanism by which photolysis of the Z,Z-isomer of bilirubin changes it into the E,E-isomer, and offer an explanation of why this change may occur.
• Task 5. Write a brief note to the publishers of Lenninger’s biochemistry text and The Merck Index indicating what is wrong with the representation(s) of bilirubin that appear in their publications and suggesting the corrections that should be made to subsequent editions of these books.

Teaching Notes

This case study was prompted by a report that appeared in the recent chemical literature (Ritter 2003). Ritter’s work pointed out errors in the structural formulas of the hemoglobin metabolic decomposition product bilirubin that appear in an important recent literature article (Baranano et al. 2002), in a major biochemistry textbook (Nelson and Cox 2000), and even in the venerable reference standard, The Merck Index (Budavari 1996). The error apparently is propagated by uncritical acceptance of a literature reference source that is copied and passed on.

This case is designed for use in introductory organic chemistry and introductory level biochemistry courses. It demonstrates to students that the technical literature is not without flaws and shows them the importance of checking critical data in a second source. My experience has been that undergraduate students are often quite surprised that errors can and do exist in the scientific literature.

Additionally, the case reviews several important concepts that are introduced in introductory organic chemistry courses and subsequently used in biochemistry courses. It also demonstrates the importance of geometric isomerism in determining the properties of biological molecules and requires students to assign priorities using the Cahn-Ingold-Prelog system and to use these priorities to assign Z-/E- designations to the configuration about double bonds in a complex biochemical system. The case also asks them to consider the mechanism that characterizes the photochemically induced interconversion of geometric isomers.

This case is best presented to students working in four-person learning teams. I give the case to students in the class before the one in which it is to be discussed. I also allow students to gather in their teams for approximately 5 minutes at the beginning of the class in which the case is to be considered. This allows the teams to exchange viewpoints on any problematic issues they uncover in the case.

At the end of this time, I give a brief, three-question, multiple-choice reading quiz. I tell students in advance that this quiz will be given, that it will focus on major points made in the case, and that they will be allowed to use notes that they have taken while reading the case. The notes must be handwritten on a 3 x 5 card. This practice helps to ensure that each student will read the case carefully before class.

A typical reading quiz for this case is as follows:

• Hemoglobin is a decomposition product of bilirubin. True or False?
• Phototherapy involves the use of light to convert bilirubin into hemoglobin. True or false?
• Which bilirubin isomer is formed in the phototherapy process? a) E,E- b) Z,Z- c) Z,E- d) E,Z-

At this point, the learning teams are ready to turn their attention to the completion of the case assignment. Working cooperatively in their teams, students prepare their responses to each of the tasks they are assigned at the end of the case. Their responses should be similar to those given in Figure 5.

The fact that errors exist in the technical literature is both surprising and interesting to students. They respond quite enthusiastically to the challenge of correcting these errors because this gives them the chance to use their newly acquired knowledge to correct errors made by seasoned professionals. Correcting the errors involving bilirubin’s structure affords students a challenge that they undertake with great enthusiasm, and one that students in a second-semester organic chemistry course are well prepared to meet.

Frank J. Dinan is a professor in the Department of Chemistry and Biochemistry at Canisius College, Buffalo, NY 14208; e-mail: dinan@canisius.edu.

References

Baranano, D.E., M. Rao, C.D. Ferris, and S.H. Snyder. 2002. Biliverdin reductase: A major physiologic cytoprotectant. Proceedings of the National Academy of Science, U.S.A. 99(25):16093–16098.
Budavari, S., ed. 1996. The Merck Index, 12th Edition. Whitehouse Station, N.J.: Merck and Company.
Fischer, H., F. Pieninger, and O. Weissbarth. 1941. Uber die konstitution des bilirubin und die bilirubinoide. Farbestoffe Hoppe-Seylers Zeitschrift F. Physicalishe Chemie 268:197–226.
Lemberg, R., and J.W. Legge. 1949. Hematin Compounds and Bile Pigments. New York: Interscience.
Nelson, D.L., and M.M. Cox. 2000. Lehninger Principles of Biochemistry. New York: Worth Publishers.
Ritter, S.K. 2003. Mind your E’s and Z’s: Due diligence by chemists is needed to prevent propagation of structural and other errors. Chemical and Engineering News 81(26):29.

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